One of the major dilemmas that an obstetrician frequently encounters in his daily practice is whether or not to deliver a woman in preterm labor (onset of labor at .ltoreq.37 weeks of gestational age), or to deliver or not a woman with some underlying disease that could be alleviated by terminating the preterm pregnancy. If the baby is delivered and the lungs of the baby are not mature or are borderline, the baby will develop Respiratory Distress Syndrome (or "RDS") which can result either in fetal death or long-lasting periods of repeated respiratory difficulty. The prevalence of RDS when the gestational age is below 31 weeks is 60%, but drops to 1% by a gestation of 36 weeks. It is estimated today that in the metropolitan Boston area alone the number of patients that need to be tested for the assessment of fetal lung maturity (or "FLM") is about 4,000 per year.
Immature fetal lungs lack an adequate surfactant layer which normally lines the alveoli and helps to keep the alveoli open after exhalation. The quantity of phospholipids generally in amniotic fluid, and of dipalmitoyl phosphatidyl choline (or "DPPC") in particular, has been correlated with the amount of surfactant lining the alveoli and with the degree of fetal lung maturity. Phosphatidyl choline (or "PC") fractional species represent nearly 80 percent of the surfactant phospholipid varieties in the fetal lung (Clements, Am. Rev. Resp. Dis, 101:984 (1970); and dipalmitoyl phosphatidyl choline (or "DPPC") constitutes about 60 percent of the fetal lung phosphatidyl choline species fraction. Other PC fraction species include 1-palmitoyl, 2-palmitoleoyI-PC (20%); 1-palmitoyl, 2-oleoyl-PC (10%); and other minor PC varietal species (10% ). The remaining lung surfactant phospholipid components also include phosphatidyl inositol, phosphatidyl ethanolamine, sphingomyelin and phosphatidyl serine. Interestingly enough, the second major phospholipid of lung surfactants is phosphatidyl glycerol, comprising more than 10 percent of the mature surfactant in the lining of the lung (Pleger and Thomas, Arch. Intern. Med, 127:863 (1971); Hallman and Gluck, Biochem. Biophys. Res. Commun. 60:1 (1974).
A number of different assays and testing techniques have been developed and used as means for estimating fetal lung maturity-with markedly different degrees of success. Some of these conventionally known assay methods employ thin layer chromatography (or "TLC") as a requisite part of the assay. Nevertheless, despite the inclusion of TLC within these assays, and notwithstanding the improvements in the techniques of thin layer chromatography in the last decade, this chromatographic technique was viewed previously and remains seen today primarily as an ancestral technique and an undesirable mode of analysis for the medical field generally and the clinical environment in particular. This disinterest, reluctance and disfavor is intrinsically revealed within the range and diversity of publications reporting and describing these assays.
For instance, in 1971 a test for the assessment of FLM was introduced by Gluck and his associates [Am. J. Obstet. Gynecol. 109:440 (1971)]. This test measures the ratio of lecithin to sphingomyelin, phosplolipids which are typically present in amniotic fluid and detected by thin layer chromatography (TLC). Subsequently, in 1976, Hallman et al. introduced the determination of phosphatidyl gylcerol (PG) in amniotic fluid by one-dimensional TLC as another test for the assessment of FLM [Hallman et al., Am. J. Obstet. Gynecol. 125:613(1976)]. A few years later, Kulovich et al. introduced the analysis of phosphatidyl glycerol (PG) in amniotic fluid using two-dimensional TLC [Kulovich et al., Am. J. Obstet Gynecol. 135:57 (1979)]. Surprisingly, since first introduced by Gluck and Hallman, TLC analysis of suffactant-associated phospholipids in amniotic fluid has become the "gold standard" for the assessment of fetal lung maturity. An L/S (Lecithin/Sphingomyelin) ratio value of .gtoreq.2 or the detected presence of phosphatidyl glycerol (PG) in amniotic fluid are considered indicative of a mature fetal lung. Also, the term "lecithin" has become synonymous with and representative of a range of different phosphatidyl cholines (PC) generally; and today designates a variety of related species that differ from each other in their fatty add residue chain lengths and/or degree of unsaturation of the fatty acids sterified to the sn-1 and sn-2 position of the glycerol moiety commonly shared among them. Despite these developments, such TLC assay methods are viewed today as cumbersome at best.
Another overwhelming problem, however, remained in that none of these assays were suitable for the assessment of FLM in samples contaminated with blood, or with meconium, or for amniotic fluid samples obtained from the vaginal pool. In 1979 an alternative test was introduced by Torday et al. for the analysis of disaturated phosphatidyl choline species (or "DSPC") from amniotic fluid [N. Engl. J. Med. 301:1013 (1979)]. In this publication, the DSPC test was introduced as a method for the assessment of FLM in amniotic fluid samples contaminated with either blood or meconium. The cut-off value used in the Torday et al. assay of DSPC was 5 .mu.g/mL; values above 5 .mu.g/mL were considered mature fetal lung tissues. This DSPC test is based on the method originally described by Mason et al. for the isolation of disaturated phosphatidyl choline species using osmium tetroxide [J. Lipid Res. 17:281 (1976)]. A further adaptation of this method was also used to measure the concentration of DSPC species in rhesus-monkey amniotic fluid as a function of gestational age. It was found that the increase in amniotic fluid DSPC species correlated with lung maturity in the fetus.
A number of deficiencies and problems, however, exist in this DSPC assay method: The test measures total disaturated lecithins which includes a variety of different chemical entities in addition to DPPC; it uses low-resolution TLC; it requires relatively high volumes of amniotic fluid samples (&gt;1 mL); it is a time-consuming test (4 to 5 h); and it acquires the use of highly toxic reagents (i.e., osmium tetraoxide and carbon tetrachloride). All of these flaws are substantive obstacles.
The historical reluctance and disfavor shown towards assays employing thin layer chromatography also lead to a continuing interest and desire, particularly by commercial pharmaceutical companies, for less complicated assay systems and less rigorous detection techniques for assessing fetal lung maturity. Thus by 1976, a new test for the assessment of FLM was introduced based on the analysis of membrane-bound vesicles in amniotic fluid by fluorescence polarization, as first described by Schinitzky et al. [Br. J. Obstet. Gynaecol. 83:833 (1976)]. In this method, a fluorophore is added to the amniotic fluid test solution containing the vesicles and the fluorophore is allowed to react with the vesicular membranes. Subsequently, when excited by plane polarized light of an appropriate wavelength, the fluorophore will emit its characteristic radiation with the same polarization as the incident light--if the molecule has not rotated with the vesicular membranes. In general, however, the angle of the emitted polarization light relative to that of the exciting light depends on how far the fluorescent molecule has interacted and rotated in the time between absorption and emission. Thus, the more fluid the vesicular membrane, the greater is the degree of rotation-resulting in relatively unpolarized fluorescent light in very fluid membranes and relatively more polarized light in less fluid membranes.
The early 1980's saw the general introduction of fluorescence polarization immunoassays for therapeutic drug monitoring determinations, with nearly every major medical center obtaining an optical instrument for this purpose such as the Abbott AMX-TDx Analyzer (Abbott Laboratories, Irving, Tex.). These instruments are highly automated and are now very well established in the clinical laboratory; and the fluorophore NBD-PC (N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]-phosphatidylchlorine) has been adapted for these instruments in a commercially available method [Tait et al., Clin. Chem. 3:2:248 (1986); Foerder et al., Clin. Chem. 32255 (1986)]. Although considerable controversy remains today as to the precise mechanism producing the observed results, this method yields results that correlate well with other measures of FLM; and, in a prospective study, compared favorably with the L/S ratio standards [Tait et al., Clin. Chem. 33:554 (1987)]. The method appears to have been quickly adopted by the medical and clinical community; and, since the test can be carried out with the AMX analyzer as a simplified, high volume, and low cost assessment of FLM, some obstetricians have been persuaded to use it as a more convenient test methodology than any of the TLC assays.
Another important test that is currently in use in hospitals across the country is the PG agglutination test which is based on the slide agglutination test of Garite et al. [Am. J. Obstet. Gynecol. 147:681 (1983)]. The results obtained in this study are typically compared to those data obtained by analysis of the L/S ratio, of phosphatidyl glycerol determination by TLC, and by fluorescence polarization for the assessment of FLM. The sensitivity of the PG agglutination test was judged to be near 100% and yielded a determined specificity of 65%. Test sensitivity is defined as the ability to predict FLM; test specificity is defined as the ability to predict the absence of FLM. In comparison, analysis by fluorescence polarization had a sensitivity of 100% and a specificity of 60% (40% falsely immature results).
More recently, in 1989,, Craig et al. introduced an ultrasensitive phosphatidyl glycerol (PG) detection kit based on the method described previously by Garite et al. [Am. J. Obstet. Gynecol. 160:298-303 (1989)]. This assay was said to have a lower limit of detection of 0.51 .mu.g of PG/mL in amniotic fluid for a positive test; and the analysis could be performed within 20 to 30 minutes. Note that the original test kit described earlier by Garite et al. had a lower limit of detection of 2 .mu.g/mL for a positive test. In their report, Craig et al. applied the ultrasensitive immunoreaction kit to the analysis of amniotic fluid obtained from the vaginal pool following premature rupture of membranes--i.e., samples contaminated with blood or meconium or patients with diabetes. Despite the lower threshold of PG detection necessary for a positive result in this new ultrasensitive test, the investigators concluded that the concordance rates were not significantly increased as compared to the results obtained by Garite et al. using the original kit. However, the data obtained using either of these kits have to be evaluated with caution in those situations where the samples are contaminated with meconium or obtained from the vaginal pool due to bacterial and/or human sperm contamination which have been shown to contain significant amounts of PG. Despite these flaws, this test is currently commercially available as the "Amniostat-FLM" test (Irvine Scientific, Calif.).
A number of other investigators have published a range of other assays methods and techniques for assessing fetal lung maturity. These have included the following systems: measurement of surfactant lecithin in sheep amniotic fluid [Ogawa, J. Exp. Med. 300:112 (1972)]; foam stability [Clements et al., N. Eng. J. Med. 286:1077 (1972)]; surface tension [Goldkrand, et al., Am. J. Obstet. Gynecol. 128:59 (1977)]; ratio of palmitic to stearic acid [Schirar et al., Am. Obstet. Gynecol. 121:653 (1975)]; surfactant apoprotein [King et al., J. Appl. Physiol. 39:735 (1975)]; and dipalmitoyl lecithin [Ogawa, Biol. Neonate. 28:18 (1976)]. With the possible exception of the amniotic fluid-foam-stability test, these methods have been difficult to use routinely or are not more reliable than the L/S ratio test or the analysis of PG by TLC, which thus remain as the gold standard for the assessment of FLM.
Overall, therefore, there remains today a continuing and long-standing need for a high-resolution, accurate and precise, and reliable assay by which to assess the maturity of fetal lung tissue. Moreover, recognizing the frequency with which amniotic fluid samples are contaminated with other living cells and body fluids, it is most desirable that such an assessment method not be markedly altered or meaningfully influenced by the presence of contaminants in the sample. Were such an assessment protocol developed, it would be recognized generally by persons in this field as a major advance and improvement over presently known tests.